Ras is one of the most highly validated targets for cancer drug discovery; however, the discovery of potent inhibitors of Ras has been difficult due to a lack of suitable binding pockets on the surface of Ras. The present inventors have discovered a binding pocket on the SOS protein, as part of the Ras:SOS:Ras complex, and small molecules that bind to this pocket and alter Ras activity in biochemical and cell-based experiments. These compounds are useful for treating cancer.
Without being bound by theory or mechanism, the Ras family of small GTPases function as molecular switches, cycling between inactive (GDP-bound) and active (GTP-bound) states to relay cellular signals in response to extracellular stimuli. The activation of Ras is tightly regulated by guanine nucleotide exchange factors (GEFs), such as Son of Sevenless (SOS), which catalyze nucleotide exchange, and GTPase-activating proteins (GAPs), which aid in GTP hydrolysis. Upon activation, GTP-bound Ras exerts its functions through protein-protein interactions with effectors such as Raf kinase and phosphoinositide 3-kinase to promote cell growth and survival.
Aberrant activation of Ras by increased upstream signaling, loss of GAP function, or oncogenic mutation results in the deregulation of cellular signals in cancer. Indeed, aberrant Ras signaling plays a role in up to 30% of all human cancers, with the highest incidence of Ras mutations occurring in carcinomas of the pancreas (63-90%), colon (36-50%), and lung (19-30%). Active Ras endows cells with capabilities that represent many of the hallmarks of cancer, including the ability to proliferate, evade programmed cell death, alter metabolism, induce angiogenesis, increase invasion and metastasis, and evade immune destruction. Importantly, inactivation of oncogenic Ras has been shown to be a promising therapeutic strategy in in vitro and in vivo models of cancer.
Embodiments of the present invention include compounds that increase the rate of SOS-mediated nucleotide exchange on Ras by binding to a functionally relevant, chemically tractable pocket on the SOS protein, as part of the Ras:SOS:Ras complex. High resolution X-ray co-crystal structures reveal the location of the binding pocket in the CDC25 domain near the catalytic site of SOS, adjacent to the Switch II region of Ras, and provide a detailed understanding of protein-ligand interactions. Mutational analyses confirmed the functional relevance of this binding site and showed it to be essential for compound activity. Perturbation of Ras signaling in HeLa and other cancer cells with these molecules demonstrates their ability to alter Ras activity in the setting of full-length proteins. The present invention is a new approach for targeting Ras signaling and provides compounds can be used to treat tumors.
Early attempts to inhibit Ras-driven tumors have focused on disrupting the posttranslational modification and localization of Ras or inhibiting Ras effectors. In contrast, embodiments of the present invention include that activate nucleotide exchange by binding to a hydrophobic pocket on the SOS protein, as part of the Ras:SOS:Ras complex. Further, these compounds perturb Ras signaling in cancer cells and kill cancer cells. This discovery represents a new approach to alter Ras activity.
Compound-mediated activation of SOS-catalyzed nucleotide exchange of the present invention is novel compared to previously reported mechanisms that increase nucleotide exchange. Mechanisms involving chelation of the divalent magnesium ion, destabilization of bound nucleotide, or activation via the allosteric Ras binding site on SOS were inconsistent with the results obtained in in vitro nucleotide exchange assays. In contrast, the present inventors discovered compounds of the present invention that increase nucleotide exchange by binding to a hydrophobic pocket on the SOS protein, as part of the Ras:SOS:Ras ternary complex.
Based on in vitro biochemical studies, the present inventors discovered that treatment of cells with compounds of the present invention resulted in diverse set of cellular responses. Indeed, Ras-GTP levels increased following the treatment of HeLa and other cancer cells with the compounds of the present invention, consistent with the observed increase in nucleotide exchange activity. Treatment of HeLa and other cancer cells would result in an increase of in downstream MAPK pathway signaling at low doses, but a decrease in MAPK signaling at higher doses. Further, downstream PI3K pathway signaling decreases in a dose-responsive manner. The present inventors thus discovered that rapid SOS-mediated activation of Ras, in the absence of other cooperative signal inputs, perturbs both MAPK and PI3K signaling.